The project focuses on the elucidation of molecular mechanisms by which the immunologically important NF-kappaB transcription factors can be activated. In addition to leading to a better basic understanding of signaling processes, identification of the molecular components of signaling pathways will also reveal potential targets for therapeutic intervention to block activation of NF-kappaB in various diseases. Inflammatory diseases, for example, are often driven by the undesirable activation of NF-kappaB. In addition, expression of the human immunodeficiency virus (HIV) and other clinically relevant viruses depends in large part on activation of NF-kappaB, making these transcription factors and their regulators potential targets for controlling the spread of HIV and other viruses. NF-kappaB transcription factors are normally retained in the cytoplasm by association with the inhibitory IkappaB proteins. Signals directly or indirectly related to pathogens or stress lead to phosphorylation and then proteolytic degradation of the inhibitor, thereby releasing the NF-kappaB factors to translocate to the nucleus and carry out their functions. Additional regulation is imposed via direct or indirect effects of signals on the transactivation activity of these factors once they have entered the nucleus. The present project is concerned with the delineation of several signaling paths that lead to phosphorylation and subsequent ubiquitin-dependent proteasomal degradation of the IkappaB inhibitors as well as those paths that stimulate transactivation. We describe an association of the adaptor-protein TANK with IKKs via NEMO. Since TANK can also associate with IKKepsilon and TBK1, two kinases structurally related to the IKKs, TANK provides a link between these kinases and the IKK complex. IKKepsilon and TBK-1 have been shown to be activated by double-stranded viral RNA sensed by Toll receptors, making it likely that TANK is critical to the NF-kappaB activation via viruses. In addition to the dissection of pathways that lead to the activation of NF-kappaB via the classical route, namely the induced degradation of small IkappaBs, we have also begun to dissect an alternative pathway of activation of NF-kappaB. This second pathway involves processing of the p100 precursor form of NF kappaB2 to the p52 form, which in particular leads to activation of p52/RelB heterodimers. Based on analyis of B cell defects in NF-kappaB2 knockout mice, we have identified the BAFF as a physiologic inducer of this second activation pathway. BAFF is a member of the TNF family of ligands and specifically targets B cells. We have determined that this second pathway of activation of NF-kappaB depends on the IKKalpha subunit of the IKK complex and on the NF-kappaB inducing kinase NIK, but that it does not require the IKK subunits NEMO or IKKbeta. Therefore the second pathway is independent of the classical IKK complex. We have now shown that the second pathway is also activated by stimulation of the lymphotoxin beta receptor on stromal cells. This discovery begins to explain some of the stromal defects observed in NF-kappB2 knockout mice. The lymphotoxin beta receptor stimulation on stromal cells could be shown to first activate the classical pathway and then the seondary pathway. This leads first to activation of p50/p65 (RelA) complexes, which then fade as the induced IkappaBalpha again represses these complexes. As p50/p65 fades, increasingly NF-kappaB activity consists of RelB complexed with p50 or p52. p100 NF-kappaB2 is the the primary inhibitor of RelB and this inhibitor is specifically degraded in the second pathway of activation of NF-kappaB, leaving RelB free to complex with p50 or p52 and enter nuclei. This research demonstrates how a single signal can lead to activation of qualitatively different NF-kappaB transcription factors, and thus elicit distinct genetic programs over time.